1. Field of the Invention
High molecular weight proteins have drawn increasing attention in recent years as knowledge of their functions and their significance in medical diagnoses has grown. One such protein is ferritin, which is the main form of stored iron in tissue. The liver is the most important site in which this form of iron is found. Also, in some pathological conditions such as overload of iron by blood transfusions, inflammation, and certain tumors, changes in the ferritin content of tissues are found. Measurement of serum ferritin reflects the amount of total iron stored in the body.
The need for sensitive and efficient assays for high molecular weight proteins such as ferritin has grown. To this end, various immunoassay techniques have been applied. The large size of these proteins, however, has heretofore limited the number of immunoassays which could provide a detectable signal. In particular, enzyme immunoassays have been limited to cumbersome procedures involving progressive binding reactions and phase separation. This is a serious limitation since enzyme immunoassays have the advantage of permitting spectrophotometric determinations and the potential of offering high sensitivity due to rapid substrate turnover rates which amplify the signal. A sensitive enzyme immunoassay is therefore needed which will permit the determination of large proteins in a simple and efficient manner.
2. Description of the Prior Art
Solid phase sandwich enzyme immunoassays for ferritin, a protein with a molecular weight of aproximately 450,000 daltons, are disclosed in Theriault et al., Clin. Chem., 23/11:2142-2144 (1977); Fortier et al., Clin. Chem., 25/8:1466-1469 (1979); Conradie et al., S. Afr. Med. J., 57:282-287 (1980); Page et al., Scand. J. Clin. Lab. Invest., 40:641-645 (1980); Anderson et al., Clin. Chim. Acta., 116:405-408 (1981); and Linpisarn et al., Ann. Clin. Biochem., 18:48-53 (1981).
Immunoassays for ferritin which do not involve enzymes include two-site immunoradiometric assays as disclosed in Addison et al., J. Clin. Path., 25:326-329 (1972) and Miles et al., Anal. Biochem., 61:209-224 (1974), competitive radioimmunoassays as disclosed in Porter, J. Lab. Clin. Med., 83:147-152 (1974) and electroimmunoassays as disclosed in Carmel et al., Anal. Biochem., 85:499-505 (1978) and Laurell, Anal. Biochem., 15:45-52 (1966).
Enzyme coupled immunoassay of insulin using m-maleimidobenzoyl N-hydroxysuccinimide ester to conjugate .beta.-D-galactosidase with insulin was described by Kitagawa et al., J. Biochem., 79:233-236 (1976). Bifunctional reagents for cross-linking various proteins are disclosed in Wold, Methods Enzymol., 25:623-651 (1972). Yoshitake et al., J. Biochem., 101:395-399 (1979) discussed conjugation of glucose oxidase and rabbit antibodies using the N-hydroxysuccinimide ester of N-(4-carboxycyclohexylmethyl)maleimide and also conjugation of antigens and antibodies with .beta.-galactosidase employing o-phenylenedimaleimide [N,N'-(1,2-phenylene)-bismaleimide]. Copending application U.S. Ser. No. 258,848 filed Apr. 29, 1981, now issued U.S. Pat. No. 4,423,143, discloses ligand-.beta.-D-galactosidase conjugates for enzyme immunoassays.